The rapid determination of trace metals in biological and environmental systems is increasingly important in identifying potential hazards and preserving the public health. The toxicity of certain metals such as lead is well-known. The absorption of even trace amounts of lead can cause severe damage to human organs. The numerous and widespread sources of lead in the environment, including the food supply, compounds the problems of screening affected groups.
It is generally recognized that lead poisoning occurs in children at blood levels as low as 10-15 ug/dl. Lead contamination of environmental sources such as water, dust and soil require identification at even lower levels. To measure these amounts, the analytical techniques must be sensitive, contaminant-specific, and reliable.
An early technique for identifying trace metals in biologic systems is described by J. Pierce et al., "Lead, Chromium, and Molybdenum by Atomic Absorption, Arch. Environ. Health, 13: 209 (1966). This measurement technique requires extraction of the lead from blood samples by precipitating the blood proteins with trichloroacetic acid and complexing the lead with a chelator under acidic conditions. The metal complex was then analyzed by atomic absorption. The skill and expensive instrumentation required by this technique limits its applicability for detecting lead contamination.
The use of d-aminolevulinic acid dehydratase (ALAD) activity in red blood cells to determine exposure to environmental lead is described by A. Berlin, et al., "European Standardized Method for the Determination of d-Aminolevulinic Acid Dehydratase Activity in Blood," Z. Klin. Chem. Klin. Biochem., 12 Jg. 1974, S. 389-390. The assay entails incubation of the enzyme with excess d-aminolevulinic acid (ALA). The porphobilinogen (PBG) which is formed within a fixed time is mixed with modified Ehrlich's reagent, and the color developed is measured photometrically against a blank. The quantity of PBG produced is a measurement of the ALAD activity and corresponds to the low levels of lead exposure.
A method of colormetric determination of ALAD is presented by S. Sassa, "Delta-Aminolevulinic Acid Dehydratase Assay," Enzyme, 28:133-145 (1982). The effect of reducing agents like dithiothreitol (DTT) on the enzyme activity and its use for the detection of subclinical lead poisoning by assaying the enzyme activity in erythrocytes is disclosed.
Two articles, each entitled "Purification and Properties of d-Aminolevulinate Dehydrase from Human Erythrocytes," first published by P. Anderson, et al., J. Biol. Chem., 254:6924-6930 (1979) and subsequently by P. Gibbs, et al., Biochem J., 230:25-34 (1985), disclose assays demonstrating lead as a noncompetitive inhibitor of ALAD activity. The incubation mixtures contained DTT, ALAD and ALA in a buffer solution. The incubations were terminated by the addition of TCA containing HgCl.sub.2. The solution was centrifuged and the supernatant was added to modified Ehrlich's reagent in acetic acid and HClO.sub.4. The colored complex formed with PBG was measured spectrophotometrically.
A similar assay measuring the activity of ALAD after exposure to lead containing samples is disclosed in a published PCT application WO 93/01310 to Silbergeld. The application suggests utilizing other well-know methods like conjugating or attaching a label to either the substrate or product and quantifying the amount of labeled material present after a defined reaction period. Another approach suggested uses a known antibody that binds specifically to unoccupied lead binding sites of ALAD.
A significant problem in using an ALAD assay is the poor lead recovery in the preparation of a sample. For an ALAD assay a sample of whole blood is typically pretreated with acid to release lead from the red blood cells and to precipitate sample proteins. The pretreated sample is then neutralized before the enzyme is added for incubation. Usually, the recovery of lead is less than the lead-spiked water control sample.
Effective screening of potentially affected people or sources demands an analytical technique which is conveniently used, inexpensive and convenient both as to the sample size and administration of the test. For early detection, contamination prevention, or corrective treatment, the time interval between sampling and results is preferably minimal. Although the lead assay using the enzyme ALAD has proven useful, there is a need to enhance the sensitivity and accuracy. The present invention provides enhancing reagents and methods which improve the sensitivity of the assay by recovering more of the lead in the sample during pretreatment and neutralization makes it available to inhibit the activity of the ALAD enzyme.